Identification of anticoagulants in a sample

ABSTRACT

The present invention is directed to a novel method of determining inhibitors of proteolytically active coagulation factors, referred to herein as anticoagulants, in a sample, in particular the qualitative detection of direct thrombin and factor Xa inhibitors in a sample. The method of the present invention allows a qualitative determination of the nature anticoagulants present in a sample. This can be achieved with only one coagulation-based test. The method can be used in a test kit, including a point-of-care system.

The present invention is directed to a novel method of determininginhibitors of proteolytically active coagulation factors, referred toherein as anticoagulants, in a sample, in particular the qualitativedetection of direct thrombin and factor Xa inhibitors in a sample. Themethod of the present invention allows a qualitative determination ofthe nature anticoagulants present in a sample. This can be achieved withonly one coagulation-based test. The method can be used in a test kit,including a point-of-care (POC) system.

The so-called coagulation cascade as part of the secondary hemostasisconsists of the contact activation pathway (also known as the intrinsicpathway) and the tissue factor pathway (also known as extrinsic pathway)leading to fibrin formation. In the tissue factor pathway, damagedtissue exposes tissue factor which activates factor VII to its activatedform VIIa. Tissue factor and factor VIIa form a complex which activatesfactor X at the common pathway. In the contact pathway, negativelycharged surfaces are exposed to the action of factor XII andprekalikrein in the blood. The activated factor XIIa activates factor XIto factor XIa. Factor XIa activates factor IX to the activated form IXa.Factor IXa, Villa, phospholipids and free calcium ions are required forthe formation of the tenase complex, which activates factor X to becomefactor Xa. The formation of the prothrombinase complex is performed viaaction of factors Xa, Va, phospholipids and free calcium ions. Saidprothrombinase complex activates the formation of thrombin fromprothrombin.

For the past 50 years vitamin K antagonists (VKAs) were the onlyavailable therapeutic solution for long-term anticoagulation in patientswith chronic thromboembolic risk. VKA treatment requires regularmonitoring which represents a major disadvantage for the patient.

In many situations including short-term treatment, different strategiesfor rapid anticoagulation are necessary. One strategy for attaininganticoagulation in patients is the direct or indirect inhibition ofactivated coagulation factors using heparins and heparinoids, whichrequire antithrombin factor III or heparin cofactor II from plasma,direct natural or synthetic inhibitors of factor IIa and Xa. Examplesare heparins, either unfractionated heparin (UFH), the sodium salt orlow molecular weight heparins (LMWHs), including but not limited toenoxaparin or its sodium salt (marketed as e.g. Lovenox®, Clexane®,Xaparin®), dalteparin or its sodium salt (marketed as e.g. Fragmin®),tinzaparin or its sodium salt (marketed as e.g. Innohep®), nadroparin(marketed as e.g. Fraxiparin®), certoparin (marketed as e.g.Sandoparin®, Mono-Embolex®), reviparin (marketed as e.g. Clivarin®).

Other thrombin inhibitors include hirudin, a natural polypeptide of 65amino acids, which is used as therapeutic agent in the form ofbivalhirudin (marketed as e.g. Angiox®) or lepirudin (marketed as e.g.Refludan®); argatroban, a derivative of arginine (marketed as e.g.Argatra®); or the tick anticoagulants.

Fondaparinux or its sodium salt (marketed as e.g. Arixtra®) is known asindirect factor Xa inhibitor, which is a synthetic pentasaccharide, withsome advantages over heparins or hirudins.

Except for the VKAs, all these drugs have to be administered parenteral,i.e. via infusion, injection or implantation, which makes them mostlyonly used for medical treatment in hospitals, e.g. in connection with amedical surgery.

Since 2000, so-called new or direct oral anticoagulants (NOACs/DOACs)have been developed to alleviate the need for recurrent monitoring andfor better handling outside the hospital environment. These NOACs playan increasingly important role in the prevention and/or treatment ofcardiovascular or thromboembolic disorders. There are currently threeNOACs (rivaroxaban, apixaban and dabigatran) in the market and new onesare expected in the near future.

NOACs act as direct anticoagulants, i.e. inhibitors of the centralprocoagulatory factors of the blood coagulation system. They includeinhibitors of factor IIa (thrombin), such as dabigatran or dabigatranetexilate (marketed as e.g. Pradaxa®, Prazaxa®, Pradax®) and inhibitorsof factor Xa, such as rivaroxaban (marketed as e.g. Xarelto®), apixaban(marketed as e.g. Eliquis®), edoxaban (marketed as e.g. Lixiana®),betrixaban or otamixaban.

There are several standard assays available to measure the effect ofcoagulation and/or the concentration of such drugs in a sample such asblood or plasma. These coagulation-based tests consist of a reagentwhich triggers the coagulation cascade in a sample such as, e.g., bloodor plasma, in order to induce clot formation. The time required for clotformation in said sample (so-called clotting time; CT) is measured by anoperator. The presence of an anticoagulant in said sample can in certaininstances lead to a delay in the time required for coagulation dependingon the nature of the test and its level of responsiveness to aparticular anticoagulant. The skilled person knows these test methods,most of them being described in e.g. EP1240528 (incorporated herein byreference).

Measurement of the activated partial thrombin time (aPTT) and theprothrombin time (PT) are examples of readily availablecoagulation-based tests which response is affected by the presence of ananticoagulant in a sample, for example, blood or plasma. Whereas the PTor international normalized ratio (INR) is used as standard assay formeasurement of VKA levels, aPTT is normally used for measuring the levelof UFH and hirudin.

For some of the routinely used coagulation tests, such as e.g. PT oraPTT, it is known that they inefficiently respond to someanticoagulants, in particular with regards to quantitative measurement.With the prothrombinase-induced clotting time (PiCT®) assay acoagulation-based test is available with efficient response to thepresence of most anticoagulant in a sample such as, e.g., blood orplasma. More specifically, PiCT® is known to efficiently respond in aquantitative manner to the presence of all known antithrombin andanti-Xa anticoagulants (see e.g. Calatzis, Haemostasis 2000, 30:172-174;Calatzis et al., Am J Clin Pathol 2008, 130:446-454). Another testmethod, the so-called ecarin clotting time (ECT) is responding to thepresence of hirudin in a sample. An amidolytic assay is known to respondto e.g. UFHs, LMWH or fondaparinux. All these coagulation-based testsare known by the skilled in the art to be useful for analysis of theeffect of an anticoagulant but only once the presence and nature of ananticoagulant is known. (see e.g. EP 1240528 or Korte et al.,Hämostaseologie 30, 212-216, 2010).

Although routine monitoring of the level of NOACs in the blood ofpatients is not recommended by drug manufacturers, NOACs have createdconsiderable concern in hemostasis laboratories since their introductioninto clinical practice due to the lack of appropriate patient managementtools or the lack of specific antidotes. Cases of major bleeding underNOAC therapy have been already reported. The need for measuring theanticoagulant effects of NOACs is still debated among experts. There aresome clinical conditions where testing for these agents would bedesirable or even life-saving. This is extremely important in anemergency situation dealing with patients with bleeding or at risk of tobleeding when an invasive procedure is needed, in case of overdosing orif a patient has developed renal failures or the like. Thus, thephysician has to know whether and in the affirmative which drug or atleast which class of drug has been taken in order to decide on immediatemeasures, including the use of the right antidote. It is also importantto note that some assays give different results depending on theconcentration of the anticoagulant, such as NOACs in the sample. Forthis assessment, a reliable test system would be also desirable, inparticular with regards to a POC system.

Some tests exist to measure specific anticoagulant concentrations thanksto calibration curves. Since there are no established therapeutic rangesto which associate appropriate therapy to, the usefulness of thesecurves can only be demonstrated upon association to clinically relevantsituations. Thus, these tests may only be used for monitoring and onlyonce the presence of the drug is known. On the other hand, there are notests available allowing to detect the presence of NOACs in samples suchas e.g. plasma or blood to identify the nature of the anticoagulant drugif present. Adaptations of existing routine hemostasis tests, e.g.(dTT), ECT, aPTT or thrombin time (TT), aimed to assess globalcoagulation status or anticoagulant effect of other anticoagulant drugsfailed to provide optimal solution to physicians for patients treatedwith NOACs.

Thus, there is a need for a readily available, easy performable andreliable testing method which could provide information of the presenceof an anticoagulant, in particular with regards to NOACs, in a sample,within a short period of time, including use in a POC system. Furtherthere is a need for a testing method which could enable identificationof the nature of such drugs, in particular with regards to NOACs in thesample such as e.g. human blood or plasma. Particularly in the case ofNOACs, this method should provide either qualitative and quantitativeinformation to the testing person, e.g. the physician or the patienthim/herself.

Surprisingly, we have now found a method wherein the presence of ananticoagulant in a sample is qualitatively determined using a singlecoagulation-based test. Moreover, the use of said singlecoagulation-based test enables the tester to determine the presence ofvarious kinds of anticoagulants such as, for example, antithrombin oranti-Xa anticoagulants in a sample, such as, e.g., human blood orplasma. For confirmation of the results, said single coagulation testcan be used in combination with other known assays, e.g. PT or aPTT, tofurther identify the nature of an anticoagulant present in a sample, inparticular in human blood or plasma. This method is in particular usefulfor identification of NOACs in a sample.

One important feature of such test system is the PiCT® technology, inparticular the Pefakit® PiCT® test, which is sensitive to all factor Xaand factor IIa inhibiting drugs. Contrary to standard routine tests oflimited specific use, Pefakit® PICT® counts with the potential ofrelying on a single test in order to address all clinical needs relatedto assessment of anticoagulant therapies. The system has beenspecifically adapted for this purpose. PiCT® is either used as disclosedin EP 1240528 or slightly adapted for certain drugs, in particularadapted as so-called inverted PiCT® as further explained herein, whichis in particular useful for identification of NOACs. The novel andinventive method is based on a surprising overreaction or underreactionof the test to certain anticoagulants when utilized making use of aparticular methodology. Furthermore, the use of this same principle incombination with other coagulation-based tests such as, for example, butnot limited to, aPTT and/or PT, allows for furtherdetermination/confirmation of the nature of the anticoagulant present ina sample, such as e.g. human blood or plasma.

This is the first time that identification of the presence of ananticoagulant, i.e. yes/no using only one test, i.e. the PiCT® system,and identification of the nature of the anticoagulant using only onetest is possible. Furthermore, we present here for the first time asystem wherein NOACs can be identified by application of only one singletest system.

PiCT® can be used for monitoring the level of all known anticoagulantswhich are directly inhibitors of factor Xa and/or factor IIa, inparticular used for monitoring the level of UFH, LMWH, hirudin,fondaparinux, argatroban, and NOACs, preferably, dabigatran,rivaroxaban, and apixaban.

A sample as used herein includes fluids taken from a human or animal, inparticular fluids taken from humans, such as e.g. whole blood or plasmacontaining the anticoagulant to be analyzed or monitored. The presentinvention is capable of determining whether an anticoagulant is presentat all in said sample and in the affirmative, the tester can determinewhich drug or class of drug is present in the sample.

As used herein, the terms “drug” and “anticoagulant” are usedinterchangeably.

As used herein, the terms Pefakit® PiCT® and PiCT® are usedinterchangeably. Pefakit® PiCT® is a diagnostic kit available from DSMNutritional Products Branch Pentapharm, Switzerland which makes use ofthe PiCT® technology.

As described by Korte et al. (supra) or in EP 1240528, Pefakit® PICT® isa functional clotting assay based on the direct activation of theprothrombinase complex which results in a fine-tuned analytical outputdirectly proportional to the final anticoagulant activity of the factorXa or factor IIa inhibitors (see Calatzis, Haemostasis 2000,30:172-174). To perform the Pefakit® PiCT® test (referred to herein asstandard “PiCT® or PiCT® Method 1), a defined amount of PiCT® activator(=reagent 1) is added to a sample, such as, e.g., plasma. The mixture isallowed to proceed at 37° C. for a specific period of time. The Startreagent (=reagent 2) is then added to the mixture and the time requiredfor clot formation is measured. The presence of anticoagulants in thesamples dose-dependently prolongs the time to clot. Pefakit® PiCT® isadapted to laboratory instruments based on mechanical or opticaldetection systems, including but not limited to ACL TOP® family(Instrumentation Laboratory), preferably ACL TOP® 500, STA compact®series (Stago) or the BCS® XP series (Siemens).

Thus, in one aspect, the present invention comprises adding to thesample, in particular selected from human blood or plasma, the reagentsand all compounds needed for the reaction to occur, thus creating areaction mixture. This reaction mixture is the subject of detection. Thereaction mixture can be provided during the detection part of themethod, but preferably is done prior to the start of the detection.Adaptation is made with regards to the amount of sample, the amount ofreagents, the reaction time, and/or, the order of reagents to be addedas described in more detail herein.

For correct interpretation of the results, specific sets of values orreference ranges (RR) are used which are adapted to the detectionsystem, e.g. the coagulometer, and the kind of testing method, such ase.g., PT, aPTT or PiCT®. The skilled person knows how to establish theseRR for the different detection systems in connection with the testingmethod used, e.g., PT, aPTT or PiCT® testing method. In case the valuetested in a sample is out of range, the tester knows that the samplecontains anticoagulants. An example of such a measurement of RR is shownin Table 1, wherein the settings are adapted for the ACL TOP® 500(Instrumentation Laboratory).

If the measurements are performed with another instrument known to theskilled person, the RR have to be determined accordingly, i.e. specificpipetting schemes have to be designed in order to achieve highreproducibility in the sample including plasma from healthy donors andnormal plasma pool (NPP) spiked with incremental amounts ofanticoagulants. After determination of the RR, the best dose responsecurves (DRCs) for each drug have to be defined for each drug and/or eachdetection system. These procedures are known in the art.

TABLE 1 PiCT ®, aPTT and PT adaptation and RR measured on ACL TOP ® 500.Values are indicated in seconds. “Out” means out of range, i.e.extension of the clotting time due to presence of anticoagulants in thesample. For more details see text. Method RR Borderline Out PiCT ®standard 21.4-36.2  36.3-39.8  39.9-300 PiCT ® fondaparinux 32.1-68.2 68.3-75.0  75.1-300 PiCT ® dabigatran 35.1-76.6  76.7-84.3  84.4-300PiCT ® inverted 21.9-106.4 106.5-117.0 117.1-300 aPTT 25.4-36.9 37.0-40.6  40.7-300 PT  9.4-12.5  12.5-13.8  13.9-100

The present invention is in one aspect directed to the use of a clottingassay, in particular the PiCT® testing method on a detection system suchas e.g. the ACL TOP® 500 analyzer and to a method to quantitatively andqualitatively detect anticoagulants in a sample, e.g. blood or plasma.Particularly, 4 different/adapted methods based on standard PiCT® (i.e.PiCT® Method 1) have been developed to achieve the best DRCs for alltypes of anticoagulants—except for anticoagulants of the VKA type—suchas in particular for anticoagulants selected from UFH, LMWH, hirudin,fondaparinux, argatroban and NOACs including dabigatran, rivaroxaban andapixaban. Thus, for identification of drugs like fondaparinux the PiCT®Method 2 is in particularly useful, including a modified/adaptedpipetting scheme compared to the standard procedure. The same turned outto be useful for dabigatran or argatroban (=PiCT® Method 3), whereinanother modified/adapted pipetting scheme is used or for NOACs such ase.g. rivaroxaban or apixaban (=PiCT® Method 4). PiCT® Method 4 or PiCT®inverted includes that first the start reagent is added to the sample,the mixture allowed to proceed according to the manufacturer'sinstructions followed by addition of the activator, i.e. a reversedorder compared to standard PiCT® (see above). PiCT® Method 1 to 4 isdescribed in more detail in the Examples.

The 4 methods (see above) are in more detail described in the Examples.Specific pipetting schemes were designed using NPP spiked withincremental amounts of anticoagulants in order to achieve highreproducibility of the test system, which is required for drugidentification including use in a POC system. With regards to the ACLTOP® 500 analyzer, PiCT® Method 1 to 4 has been established as follows:

-   -   PiCT® Method 1: 50 μl of plasma is incubated for 180 sec with 50        μl of reagent 1 and afterwards 50 μl of reagent 2 is added and        the time to clot is recorded on the ACL TOP® 500 (i.e. pipetting        scheme: P-50/A-50/I-180/ 50-S). This method is in particularly        useful for detection of UFH, LMWH or hirudin.

PiCT® Method 2: P-75/A-65/I-180/35-S on the ACL TOP® 500 orP-80/A-65/I-180/40-Son the STA compact® or P-75/A-65/I-180/S-40 on theBCS® XP. This method is in particularly useful for fondaparinux.

PiCT® Method 3: P-90/A-70/I-180/40-Son the ACL TOP® 500 or STA compact®,or P-70/A-60/I-180/45-Son the BCS® XP. This method is in particularlyuseful for dabigatran or argatroban.

PiCT® Method 4: P-140/S-45/I160/A-40 on the ACL TOP® 500 or STAcompact®, or P-125/S-45/I-150/A-40 on the BCS® XP. This method is inparticularly useful for rivaroxaban or apixaban.

Thus, in one aspect the present invention is directed to the use ofPiCT® for identification of NOACs, in particular rivaroxaban orapixaban, in a sample, such as e.g. human blood or plasma, wherein thesample is first supplemented with the activator reagent, the mixtureallowed to proceed and finally supplemented with the start reagent.

In one aspect, the present invention is directed to a method ofmeasuring the presence of an anticoagulant in a sample, such as a fluid,in particular from human or animal, preferably blood or plasma. Thus,the physician/patient, i.e. the tester, needs to know whether or not ananticoagulant is present in e.g. a blood or plasma sample. A firstevaluation is done by standard PiCT® testing (PICT® Method 1): ameasurement of clotting times out of range, i.e. above the RR, indicatesthe presence of UFH, LMWH, hirudin, argatroban, fondaparinux ordabigatran in the sample. With regards to NOACs such as rivaroxaban orapixaban, this Method 1, i.e. the standard PiCT®, is in the RR even ifconcentrations in the range of about 300 ng/ml are present in thesample. In the case of dabigatran, the clotting times measured with thismethod are above the RR, meaning out of range.

The detection of (low) levels of anticoagulants including NOACs in asample is a further aspect of the present invention. The use ofso-called inverted PiCT® testing method (see above) turned out to be aparticular useful method for this purpose.

Examples of anticoagulants, preferably in low concentrations, which canbe detected with said method (PICT® Method 4) are selected fromdabigatran, heparins such as e.g. UFH and LMWH, hirudin, fondaparinux,argatroban as well as the NOACs, e.g. rivaroxaban or apixaban. As usedherein, a low concentration of anticoagulant means a range of 20 to 35ng/ml with regards to rivaroxaban, apixaban or dabigatran, in the rangeof 0.3 to 0.4 μg/ml with regards to fondaparinux or argatroban orhirudin, in the range of 0.3 to 0.4 IU/ml with regards to heparins, suchas e.g. UFH or LMWH.

Thus, for identification of drugs such as dabigat ran, rivaroxaban orapixaban the inverted PiCT® testing method (referred herein as PiCT®Method 4) is used as detection test.

To determine whether the NOAC in the sample is dabigatran or the like,the first test, i.e. PiCT® Method 1 resulted in clotting times out ofrange, a second test is performed, wherein the inverted ^(PiCT)®described above, corresponding to PiCT® Method 4, together e.g. with theACL TOP® 500 analyzer, can be used and a clotting time above the RR ofabout 21.4 to about 37.9, such as e.g. above about 29.6, means thepresence of dabigatran in the sample. The RR measured with dabigatran(see above) using the standard PiCT® gives results out of range, whichis in contrast to other NOACs such as rivaroxaban and apixaban. Theparticular clotting times measured for the different anticoagulants areshown in Table 2.

In one particular aspect, the present invention is directed to a methodto determine which kind of anticoagulant is present in a sample, e.g. inparticular whether the sample comprises an anticoagulant selected fromthe group consisting of UFH, LMWH, hirudin, fondaparinux, argatroban,dabigatran, rivaroxaban and apixaban, wherein the sample may be selectedfrom a fluid, such as in particular from human or animal, preferablyblood or plasma. This method basically is a combination of 4 tests,wherein first PiCT® Method 1 and 4 are performed, combined with aPTT andPT (for confirmation of the results) and the combined results, i.e.measured clotting times, are analyzed with the detection system such ase.g. the ACL TOP® 500 analyzer:

-   (1) If all 4 tests result in clotting times within the RR determined    beforehand (see e.g. in Table 1 as established for the ACL TOP® 500    analyzer), there is no drug present in the sample.-   (2) If PiCT® Method 1 is within the RR, but Method 4 gives results    out of range (such as e.g. clotting times below about 24.4 to about    30.4, such as e.g. below about 27.6, and if the clotting times    according to PiCT® Method 4 are above the RR of about 21.4 to about    37.9, such as e.g. above about 29.6 as established for the ACL TOP®    500 analyzer shown in Table 1), the sample contains NOACs selected    from rivaroxaban or apixaban. Depending on the concentration, the PT    to and aPTT are either out of RR (i.e. PT out of RR with higher    concentrations of both NOACs) or within the RR (i.e. aPTT within the    RR with normal or lower concentration of both NOACs). This is in    more detailed outlined in the Examples.-   (3) If the modified PiCT®, i.e. PiCT® Method 3, as well as PiCT®    Method 4 show clotting times out of range, the sample might contain    either dabigatran or argatroban. These results would be confirmed    with the PT and aPTT, i.e. clotting times also above RR. Only in    case of low concentration of dabigatran, the PT shows clotting times    within the RR.

(4) If the standard PiCT®, i.e. PiCT® Method 1, as well as PiCT® Method4 show clotting times out of range, the sample might contain either UFH,LMWH or hirudin. Whereas the aPTT might be also out of range for allthree anticoagulants, PT will be out of range only for hirudin, butwithin the RR for UFH, LMWH or low concentration of hirudin.

-   (5) If the modified PiCT®, i.e. PiCT® Method 2, as well as    PiCT®Method 4 is out of range, the sample might contain    fondaparinux. In this case, PT and aPTT show clotting times within    RR.

By the addition of polybrene a distinction can be done, which of theanticoagulants is really present in the sample: if after addition ofpolybrene the measured clotting times are again back to RR when usingthe PiCT® Method 1, the sample comprises heparins, such as e.g. UFH orLMWH. Alternatively, if the result is not going back to RR, the samplecomprises low concentrations of dabigat ran or hirudin. Similar resultsare obtained with NOACs like rivaroxaban or apixaban in the sample:after addition of polybrene the measured clotting times are out of rangewhen using the PiCT® Method 1 (instead of within the RR without theaddition of polybrene). However, in case of low concentrations ofrivaroxaban or apixaban in the sample, the addition of polybrene has noeffect on the clotting times. The particular clotting times measured forthe different anticoagulants are shown in Table 6, which is an exampleof results obtained in spiked plasma samples.

In one particular embodiment, the present invention relates to a methodfor quantification the effect of anticoagulants in a sample, wherein thesample may be selected from a fluid, such as in particular from human oranimal, preferably blood or plasma. In particular, the PiCT® Method 1can be used to detect the effect of hirudin or heparins such as e.g. UFHand LMWH, together with e.g. the ACL TOP® 500 analyzer. Thus, first thesample such as e.g. plasma, in particular 50 μl of plasma, is incubated,in particular for 180 sec, with the reagent 1, in particular 50 μl, andafterwards the reagent 2, in particular 50 μl, is added and the time toclot is recorded as described above. The particular clotting timesmeasured for UFH, LMWH or hirudin shown in Table 7 are examples ofresults obtained in spiked NPP samples.

If the effect of fondaparinux in a sample is to be quantified, a methodsimilar to PiCT® Method 1 is used, referred herein and in Table 3 asPiCT® Method 2, together with e.g. the ACL TOP® 500 analyzer, i.e. firstthe sample such as e.g. plasma, in particular 75 μl, is incubated, inparticular for 180 sec, with the reagent 1, in particular 65 μl, andafterwards the reagent 2, in particular 35 μl, is added and the time toclot is recorded, i.e. DRC. The particular clotting times measured forfondaparinux shown in Table 8 are examples of results obtained in spikedNPP samples.

In the case of dabigatran or argatroban, i.e. the determination of thequantitative effect of said drug in a sample, a method similar asdescribed above and referred herein and in Table 3 as PiCT® Method 3,may be used together with e.g. the ACL TOP® 500 analyzer, i.e. first thesample such as e.g. plasma, in particular 90 μl, is incubated, inparticular for 180 sec, with the reagent 1, in particular 70 μl, andafterwards the reagent 2, in particular 40 μl, is added and the time toclot is recorded. The particular clotting times measured for dabigatranare shown in Table 9, which is an example of results obtained in spikedNPP samples.

In the case of rivaroxaban or apixaban in a sample, PiCT® Method 4together with e.g. ACL TOP® 500 analyzer may be used, wherein DRC isgenerated with extrapolation of the relevant clotting times. The resultsare shown in Table 10, which is an example of results obtained in spikedNPP samples.

Evaluation if the level of NOACs in a sample are low enough to definethis as non-critical situation is a further object of the presentinvention. To do this determination, the sample may be selected from afluid, such as in particular from human or animal, preferably blood orplasma, and the clotting times measured via PiCT® Method 4 together withe.g. ACL TOP® 500 analyzer. In the case the non-critical level forrivaroxaban or apixaban have to be measured in the sample, first DRCshave to be developed and the relevant clotting times extrapolated. Theresults are shown in Table 11, which is an example of results obtainedin spiked plasma samples. For dabigatran measurement, PiCT® Method 3described above might be used, together with e.g. the ACL TOP® 500analyzer. The results are shown in Table 11 using spiked plasma samples.

The present invention comprises the following embodiments, which areonly examples and not intended to limit the scope of the presentinvention:

1. A method for the determination of the presence of an inhibitor ofcoagulation in a sample, preferably selected from blood or plasma, usingonly one coagulation-based test, in particular prothrombinase-inducedclotting test (PiCT®).

2. A method according to claim 1 to identify whether or not ananticoagulant is present in a sample, preferably selected from humans oranimals, more preferably selected from blood or plasma, usingprothrombinase-induced clotting test (PiCT®), wherein the start reagentis added to the sample before the mixing with activator reagent.

3. A method according to claim 1 to determine which kind of new oralanticoagulants (NOACs) is present in a sample, preferably selected fromhumans or animals, more preferably selected from blood or plasma, usingprothrombinase-induced clotting test (PiCT®).

4. A method according to claim 1 to determine which kind ofanticoagulant is present in a sample, preferably selected from humans oranimals, more preferably selected from blood or plasma, usingprothrombinase-induced clotting test (PiCT®) combined with aPTT and/orPT.

5. The method according to claim 4, wherein the sample comprises ananticoagulant selected from the group consisting of UFH, LMWH, hirudin,fondaparinux, argatroban, dabigatran, rivaroxaban and apixaban.

6. A method according to claim 1 for quantification the effect ofanticoagulants in a sample, preferably selected from humans or animals,more preferably selected from blood or plasma, usingprothrombinase-induced clotting test (PiCT®).

7. A method according to claim 1 for evaluation of the level of NOACs ina sample, more preferably selected from blood or plasma, usingprothrombinase-induced clotting test (PiCT®).

8. Use of a combination of prothrombin time (PT), activated partialthromboplastin time (aPTT) and prothrombinase-induced clotting test(PiCT®) for determination of inhibitors of coagulation in a sample,preferably selected from blood or plasma.

9. Use according to claim 8, wherein the inhibitors of coagulation areselected from direct thrombin and factor Xa inhibitors.

10. Use according to any one of claim 8 or 9, wherein within theprothrombinase-induced clotting test the activator can be added beforeor after the addition of the start reagent.

FIGURES

FIG. 1. DRC for the different anticoagulants using PiCT® Method 1 (FIG.1A), PiCT® Method 2 (FIG. 1B), PiCT® Method 3 (FIG. 1C), PiCT® Method 4(FIG. 1D) using the ACL TOP® 500 analyzer. The therapeutic range isindicated by the “T”, the prophylactic range is indicated by the “P”.For further details see text.

FIG. 2. Evaluation of a sample wherein fondaparinux is present. Valuesout of range are in bold, values in the RR are indicated with a greybackground color, borderline values are in Italic. The anticoagulantsare listed in the right-hand column, with UFH, LMWH, Fonda(fondaparinux), hirudin, argatroban, Dabi (dabigatran), Riva(rivaroxaban), Apixa (apixaban). NPP without any anticoagulants is usedas control. The columns are as follows: (A)=standard PiCT®, (B)=invertedPiCT®, (C)=aPTT, (D)=PT; (E)=polybrene standard to PiCT®, (F)=polybreneinverted PiCT®.

FIG. 3. Evaluation of a sample wherein either UFH, LMWH, hirudin or lowconcentration of dabigatran is present. Values out of range are in bold,values in the RR are indicated with a grey background color, borderlinevalues are in Italic. For more detail see text or legend to FIG. 2.

FIG. 4. Confirmation of results shown in FIG. 3 for UFH and LMWH. Formore detail see text of legend to FIG. 2.

FIG. 5. Evaluation of a sample wherein rivaroxaban or apixaban ispresent. For more detail see text or legend to FIG. 2.

FIG. 6. Evaluation of a sample wherein hirudin, argatroban or dabigatranis present. For more details see text or legend to FIG. 2.

FIG. 7. In order to test the method for feasibility under real-lifeconditions and to evaluate its use in a point-of-care (POC) system,individual plasma samples were tested with the combination of assays(see legend to FIG. 2). For each patient, two separate runs wereperformed. The results are shown in FIGS. 7A and 7B for patient 1 andFIGS. 7C and 7D for patient 2.

The following examples are illustrative only and are not intended tolimit the scope of the invention in any way. EXAMPLES

Stock solutions of anticoagulants were prepared in deionized water forUFH (250 IU/mL, WHO standard 07/328, NIBSC), LMWH (250 IU/mL, Fragmin®,Pfizer), and lepirudin (0.5 mg/mL, (Refludan®, Bayer HealthCarePharmaceuticals Inc) or in DMSO for rivaroxaban (0.5 mg/mL, Alsachim,France) and apixaban (2 mg/mL, Alsachim) or in 0.1M HCl for dabigatran(Alsachim), and were stored at −20° C. Fondaparinux (5 mg/mL, injectablesolution, Arixtra®, GlaxoSmithKlines) was stored at 4° C. Anticoagulantswere further diluted with deionized water and spiked in NPP preparedfrom 24 healthy donors (Universitätsklinikum Würzburg, Germany) forPiCT® measurements. The final concentrations of DMSO or HCl in NPP didnot exceed 0.15% and 0.4 mM respectively, and did not affectcoagulation.

Example 1 PiCT® Adaptations and Determination of Reference Ranges (RR)

As a starting point, the RR for four different types of anticoagulantshad to be established as described above. These have to be adapted tothe testing system and also to the kind of drug, as e.g. in the case offondaparinux (PICT® Method 2) or dabigatran/argatroban (PICT® Method 3).The results are shown in Table 2 wherein the results are given usingfresh plasma from 17 healthy donors.

TABLE 2 Establishing the RR on the ACL TOP ® 500. The clotting time (RR,borderline, out of range) is given in seconds, indicating the mean andthe lower and upper ranges (in parentheses). Method RR PiCT ® Method 127.6 (24.4-30.7) PiCT ® Method 2 40.0 (31.9-48.1) PiCT ® Method 3 44.7(38.1-51.3) PiCT ® Method 4 29.6 (21.4-37.9) HemosIL ™ aPTT 25.4-36.9 SPHemosIL ™ PT  9.4-12.5 recombiplastin

Protocols and pipetting schemes designed for ACL TOP® 500 are describedin Table 3. To assess the potential of Pefakit® PiCT® to evaluateanticoagulant effects of most common anticoagulant drugs, DRCs wereperformed using NPP spiked with increasing levels of UFH, LMWH,fondaparinux, hirudin, rivaroxaban, apixaban or dabigatran. The detailsof the different methods, i.e. PiCT® Method 1 to 4, are described in thetext.

TABLE 3 pipetting scheme for the 4 different PiCT ® Methods described inthe text, wherein “P” means plasma in μl, “A” means activator reagent inμl, “S” means start reagent in μl, and “I” means incubation time inseconds. PiCT ® methods Protocol for ACL TOP ® 500 Best DRC fit forMethod 1 P-50/A-50/I-180/S-50 UFH, LMWH, hirudin Method 2P-75/A-65/I-180/S35 Fondaparinux Method 3 P-90/A-70/I-180/S40Argatroban, dabigatran Method 4 P-140/S-45/I-160/A-40 Rivaroxaban,apixaban PiCT ® Method 4 was identified to offer best results. Itconsists of inverting the order of reagents' addition to plasma. PiCT ®is very sensitive to low levels of NOACs.

Example 2 Pefakit® PiCT® as a Tool to Identify Whether a Plasma SampleContains an Anticoagulant

PiCT® Method 4, which is an inverted PiCT®, was developed as new methodto achieve best DRCs for rivaroxaban and apixaban. After calculation ofthe RR for the different kind of anticoagulants (see Example 1) samplesof NPP spiked with incremental amounts of anticoagulants were used inthe ACL TOP® 500, wherein 45 μl start reagent was incubated with 140 μlplasma for 60 sec. The time to clot was recorded after addition of 40 μlof activator (i.e. so-called inverted PiCT®). The result is shown inTable 4, wherein the number of samples (NPP) spiked with incrementalamounts of anticoagulants (AC) is indicated (n), i.e. indicated as“treatment”. Every sample with a result starting from a certainthreshold above the RR contains an anticoagulant, which is true for alldrugs listed in the Table.

TABLE 4

identification of anticoagulants in plasma samples using PiCT^(®) Method4. The baseline means the RR in a sample without the addition of anydrug and is shown as mean and as whole range (in brackets). “Conc.”means concentration of the anticoagulant per ml of sample. Numbers inbold indicates a clotting time out of range, i.e. presence of a drug inthe sample, wherein the negative control, i.e. within RR is marked withthe grey background color.

Using this method, low amounts of all NOACs, UFH, LMWH, hirudin,fondaparinux or argatroban could be detected as shown in Table 4.

Example 3 Pefakit® PiCT® as a Tool to Determine Which NOAC is Present ina Sample

With the normal PiCT®, i.e. PiCT® Method 1, wherein 50 μl of plasma ismixed with the same amount of reagent 1 (PICT® activator), incubated for180 sec and mixed with the same amount of reagent 2 (PICT® startreagent) in a ACL TOP® 500 analyzer, the presence of NOACs cannot bedetermined in a sample. This is true even at high levels of said drugsin the sample, i.e. in the range of 300 ng/ml. Thus, in order todetermine which NOAC is present in the sample, a combination of PiCT®Method 1 and PiCT® method 4 has to be used (see above for detaileddescription). To confirm this, spiked plasma samples with differentlevels of NOACs were prepared and the clotting time measured using firstPiCT® Method 1 followed by PiCT® Method 4. The result is shown in Table5. In the case of rivaroxaban or apixaban present in a tested sample,PiCT® Method 1 is within the range (indicated by the grey backgroundcolor) whereas PiCT® Method 4 is out of range (indicated in bold). Incase of dabigat ran present in the sample, both PiCT® Method 1 (whichhad to be adapted, i.e. PiCT® Method 3) and 4 are out of range asindicated in bold.

TABLE 5

distinction between different NOACs present in a sample determined by asubsequent performance of PiCT^(®) Method 1 (PiCT^(®) Method 3 fordabigatran) and PiCT^(®) Method 4. Results within the RR are indicatedby the grey background color, results above the RR are marked in bold.For more details see text or legend to Table 4.

Example 4 Pefakit® PiCT® as a Tool to Further Determine the Nature of anAnticoagulant in a Plasma Sample

In order to address the above question, i.e. to determine the nature ofan anticoagulant in a sample, a combination of several methods has to beperformed, including PiCT® Method 1, PiCT® Method 4, aPTT (HemosIL™aPTTSP), PT (HemosIL™PT recombiplastin), and sometimes even a modified PiCT®Method 1 wherein polybrene has been added. In order to simulate this,plasma samples spiked with different levels of anticoagulants wereanalyzed using the ACL TOP® 500 analyzer. The results are shown in Table6. For each drug, a specific pattern was obtained, i.e. clotting timeswithin the RR and clotting times above the RR, depending on the usedtesting method, which is unique at least for the class of anticoagulantor even for a specific anticoagulant.

TABLE 6

scheme for determination of what kind of anticoagulant is present inspiked plasma samples. Only mean values are given. The “n” refers to thenumber of healthy donors. For more details see text and legend to Tables4 or 5.

In case of a measurement using PiCT® Method 1 and PiCT® Method 4,wherein the clotting time determined with the first method is within theRR but the time measured with the second method is above the RR, theanticoagulant present in the sample is either apixaban or rivaroxaban.Further tests have been performed, i.e. the aPTT or PT in order todistinguish between both anticoagulants: in case of rivaroxaban, withboth tests the clotting times were above the RR—but only at higherdoses, such as in the range of 200 ng or more (see Table above). If, forexample, both the results from PiCT® Method 1 and 4 are above the RR butthe aPTT and PT revealed clotting times within the RR, the drug presentin the sample is fondaparinux.

Example 5 Pefakit® PiCT® as a Tool to Quantify the Effect of anAnticoagulant

Depending on the drug used, the protocols to determine the quantitativeeffect of an anticoagulant might differ, i.e., using different testingmethods, including the ones listed in Table 1 showing the RRs for thevarious methods. If the effect of heparins or hirudins, such as e.g.UFH, LMWH and hirudin, is to be quantified, Pefakit® PiCT®with PiCT®Method 1 turned out to be useful, which is shown in Table 7A-C showingDRC in NPP samples spiked with UFH (Table 7A), LMWH (Table 7B) orhirudin (Table 7C) as measured with PiCT® Method 1 and ACL TOP® 500analyzer. Only mean values are given. For the baseline, i.e. noanticoagulant was present in the sample, the number of measurements wasn=6 with a clotting time of 24 seconds measured with PiCT® Method 1.

TABLE 7A PiCT ® Method 1 using NPP spiked with UFH and n = 4. For moredetails see text and legend to Table 4. Concentration [IU/ml] 0.1 0.20.3 0.5 0.7 0.9 1.1 1.3 1.5 1.6 Clotting 30 45 62 86 102 113 123 132 143147 time [sec]

TABLE 7B PiCT ® Method 1 using NPP spiked with LMWH and n = 4. For moredetails see text and legend to Table 4. Concentration [IU/ml] 0.1 0.20.3 0.4 0.5 0.7 0.9 1.0 1.2 1.4 Clotting 37 47 57 67 76 92 108 114 124132 time [sec]

TABLE 7C PiCT ® Method 1 using NPP spiked with hirudin and n = 4. Formore details see text and legend to Table 4. Concentration [μg/ml] 0.20.4 0.6 0.9 1.3 1.7 2.0 2.4 2.8 3.0 Clotting 41 55 69 86 113 142 158 183209 219 time [sec]

For fondaparinux, PiCT® Method 2 was determined to be useful. Examplesof results obtained with NPP samples on ACL TOP® 500 analyzer are shownin Table 8.

TABLE 8 DRC in NPP samples spiked with fondaparinux as measured withPiCT ® Method 2. Only mean values are given. The number of measurementswas n = 4. Clotting time for the baseline, i.e. no anticoagulant presentin the sample, was 39 seconds measured with PiCT ® Method 2.Concentration [μg/ml] 0.2 0.4 0.5 0.6 0.9 1.1 1.3 1.5 1.8 2.0 Clotting78 92 98 103 120 122 133 139 148 155 time [sec]

With regards to dabigatran and argatroban, the DRC was established usingPiCT® Method 3, the relevant clotting times were extrapolated with theparticular conditions as described herein. For all measurements, the ACLTOP® 500 analyzer was used making use of NPP samples spiked withincremental amounts of anticoagulant covering both average peak ortrough (see Stangier J, Clin Pharmacokinet 2008, 47:47-59; van Ryn J,Thromb Haemost 2010, 103:1116-1127; Baglin T, J Thromb Haemost. 2013 Jan24). Examples of results obtained with NPP samples spiked with eitherdabigatran (Table 9A) or argatroban (Table 9B) are presented below.

TABLE 9A DRC in NPP samples spiked with dabigatran as measured withPiCT ® Method 3. Only mean values are given. The number of measurementswas n = 4. Clotting time for the baseline, i.e. no anticoagulant presentin the sample, was 45 seconds measured with PiCT ® Method 3.Concentration [ng/ml] 5 10 50 100 200 300 400 500 600 750 Clotting 56 70122 153 190 207 220 234 245 253 time [sec]

TABLE 8B DRC in NPP samples spiked with argatroban as measured withPiCT ® Method 3. Only mean values are given. The number of measurements(“n”) was n = 4. Clotting time for the baseline, i.e. no anticoagulantpresent in the sample, was 45 seconds measured with PiCT ® Method 3.Concentration [μg/ml] 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Clotting113 145 162 179 198 204 210 232 234 240 time [sec]

Regarding rivaroxaban or apixaban, DRC was established making use ofPiCT® Method 4 with extrapolation of relevant clotting times associatedwith the particular conditions. The following are examples of clottingtimes obtained in ACL TOP® 500 analyzer making use of NPP samples spikedwith incremental amounts of anticoagulant levels covering both averagepeak and trough concentrations of rivaroxaban (see Mueck W, ThrombHaemost 2008, 100:453-461; Xu XS Br J Clin Pharmacol 2012, 74:86-97;Baglin T, J Thromb Haemost. 2013 Jan. 24) as shown in Table 10A orapixaban (see Raghavan N, Drug Metab Dispos 2009, 37:74-81; Frost C, BrJ Clin Pharmacol 2012, 75:476-487; Frost C, Br J Clin Pharmacol 2013,76:776-786) as shown in Table 10B. Only mean values are given. For thebaseline, i.e. no anticoagulant was present in the sample, the number ofmeasurements was n=4 with a clotting time of 54 seconds measured withPiCT® Method 4.

TABLE 10A PiCT ® Method 4 using NPP spiked with rivaroxaban and n = 4.For more details see text. Concentration [ng/ml] 5 10 50 100 200 300 400500 600 Clotting 71 83 118 149 191 220 248 270 278 time [sec]

TABLE 10B PiCT ® Method 4 using NPP spiked with apixaban and n = 4. Formore details see text. Concentration [ng/ml] 5 10 50 100 200 300 400 500600 750 Clotting 65 74 109 136 167 197 210 233 259 268 time [sec]

Example 6 Pefakit® PiCT® as a Tool to Evaluate if NOAC Levels are LowEnough

For answering this question with regards to rivaroxaban or apixaban, DRCwas to generated making use of PiCT® Method 4 (see above). The relevantclotting times associated with the particular conditions wereextrapolated. All data were obtained using the ACL TOP® 500 analyzer andplasma samples spiked with anticoagulant levels corresponding toconcentration in the plasma reported as wash off levels. The result isshown in Table 11.

TABLE 11 use of PiCT ® Method 4 and plasma samples spiked with differentlow concentrations of two anticoagulants, i.e. rivaroxaban and apixaban.For more details see text. PiCT ® method 4 clotting time Rivaroxaban:−20 ng/mL 70 sec (n = 3) −35 ng/mL 80 sec (n = 3) Apixaban: −20 ng/mL 45sec (n = 3) −35 ng/mL 54 sec (n = 3)

In order to evaluate the level of dabigatran in a sample, DRC wasgenerated making use of PiCT® Method 3, the relevant clotting timesassociated with the particular conditions were extrapolated. All datawere obtained using the ACL TOP® 500 analyzer and plasma samples spikedwith anticoagulant levels corresponding to concentration in the plasmareported as wash off levels. The result is shown in Table 12.

TABLE 12 use of PiCT ® Method 3 and plasma samples spiked with differentlow concentrations of dabigatran. For more details see text. PiCT ®method 3 clotting time Dabidat ran: −20 ng/mL  84 sec (n = 11) −40 ng/mL109 sec (n = 11)

1-11. (canceled)
 12. A method for identification of a drug inhibitingcoagulation-factor IIa or factor Xa in a sample, preferably selectedfrom blood or plasma, comprising the steps of: (a) establishing thereference ranges for prothrombinase-induced clotting time (PiCT®) usingreference samples with increasing concentrations of knownanti-coagulants, (b) mixing the sample to be tested with PiCT®-activatorand PiCT®-start reagent, in particular wherein the start reagent isadded before addition of the activator reagent, (c) measuring andcomparing the PiCT®; wherein the presence of an anti-coagulant in thetest sample is indicated by a PiCT® out of range.
 13. A method accordingto claim 12, using only one coagulation-based test, in particularprothrombinase-induced clotting time (PiCT®) assay.
 14. A methodaccording to claim 12 comprising prothrombinase-induced clotting time(PiCT®) assay, wherein the start reagent is added to the sample beforethe mixing with activator reagent.
 15. A method according to claim 12,wherein the anti-coagulation drug is selected from direct oralanti-coagulants (NOACs), preferably rivaroxaban, apixaban anddabigatran.
 16. A method according to claim 12, wherein PiCT® assaycombined with activated partial thrombin time (aPTT) and/or prothrom bintime (PT).
 17. The method according to claim 12, wherein the samplecomprises an anticoagulant selected from the group consisting of UFH,LMWH, hirudin, fondaparinux, argatroban, dabigatran, rivaroxaban andapixaban.
 18. A method according to claim 12 for quantification theeffect of anticoagulants in a sample, preferably selected from humans oranimals, more preferably selected from blood or plasma, usingprothrombinase-induced clotting time (PiCT®) assay.
 19. Use of acombination of prothrombin time (PT), activated partial thromboplastintime (aPTT) and prothrombinase-induced clotting time (PiCT®) foridentification of inhibitors of coagulation in a sample, preferablyselected from blood or plasma.
 20. Use according to claim 19, whereinthe inhibitors of coagulation are selected from direct thrombin andfactor Xa inhibitors.
 21. Use according to claim 19, wherein within theprothrombinase-induced clotting test the activator can be added beforeor after the addition of the start reagent.
 22. Use of a methodaccording to claim 12 in a point-of-care system.